The present invention relates in general to farnesyl protein transferase (i.e., farnesyltransferase) inhibitors. More particularly, the present invention relates to peptoid and semipeptoid peptidomimetic compounds derived from a farnesyltransferase universal recognition tetrapeptide sequence CAAX (i.e., CAAX motif, SEQ ID NO:1) and analogs thereof, and to the use of these compounds and analogs and ester derivatives thereof as chemotherapeutic agents in oncogenic as well as non-oncogenic Ras associated cancers and other proliferative diseases.
The use of peptides as drugs is limited by the following factors: (a) their low metabolic stability towards proteolysis in the gastrointestinal tract and in serum; (b) their poor absorption after oral ingestion, in particular due to their relatively high molecular mass or the lack of specific transport systems or both; (c) their rapid excretion through the liver and kidneys; (d) their undesired side effects in non-target organ systems, since peptide receptors can be widely spread in an organism; and (e) their high immunogenicity.
In recent years intensive effort has been directed towards the development of peptidomimetics (i.e., peptide analogs) that display more favorable pharmacological properties than their peptide prototypes. A peptidomimetic is a compound that, as a ligand of a receptor, can imitate or block the activity and biological effect of a peptide at the receptor level (Giannis and Kotler, Angew. Chem. Int. Ed. Engl., 1993, 32: 1244). The following requirements exist for the pharmacological properties of a peptidomimetic: (a) metabolic stability; (b) good bioavailability; (c) high receptor affinity and receptor selectivity; and (d) minimal side effects.
The native peptide itself, the pharmacological properties of which should be optimized, generally serves as the lead structure for the development of peptidomimetics. With few exceptions, linear peptides of small or medium size (&lt;30-50 amino acid units) exist in dilute aqueous solution in a multitude of conformations in dynamic equilibrium. If a receptor ligand has a biological active conformation per se, in other words, the receptor-bound conformation, then an increased affinity toward the receptors is expected and generally experienced, as compared with the flexible ligand.
From a pharmacological and medical points of view, it is in many cases desirable not only to imitate the effect of the peptide at the receptor level, as an agonist, but also to block the receptor, as an antagonist. The same pharmacological considerations mentioned above holds for peptide antagonists, but, in addition, their development in the absence of lead structures is more difficult. In most cases it is not unequivocally clear which factors are decisive for an agonistic effect and which are for antagonistic effect.
A generally applicable and successful method for the development of peptidomimetics involves formation of conformationally restricted analogs that imitate the receptor-bound conformation of the endogenous ligands as closely as possible (see, Rizo and Gierasch, Ann. Rev. Biochem., 1992, 61: 387). Investigating these analogs demonstrated their increased resistance toward proteases resulting in increased metabolic stability, as well as their increased selectivity. As a result of these properties, peptidomimetics produce less side effects (Veber and Friedinger, Trends Neurosci., 1985, 8: 392-396). The observation that in many cases only a small number (e.g., four to eight) of amino acid side chains of a peptidic ligand are responsible for recognition of the ligand by its receptor turns out to be favorable for this approach. In such cases the rest of the ligand molecule framework serves to fix the amino acids responsible for recognition, also known in the art as pharmacophores, in a specific spatial arrangement.
Compounds having a rigid conformation are then produced, and the most active structures are selected by assays analyzing the structure-activity relationship. Such conformational constraints can involve short range (local) modifications of the structure or long range (global) conformational restraints.
For example, bridging between two neighboring amino acids in a peptide leads to local conformational modifications, the flexibility of which is limited in comparison with that of a native dipeptides. Some possibilities for forming such bridges include incorporation of lactams and piperazinones. .gamma.-lactams and .delta.-lactams have been designed as "turn-mimetics"; in several cases the incorporation of such structures into peptides lead to biologically active compounds (Giannis and Kotler, Angew. Chem. Int. Ed. Engl., 1993, 32: 1244).
Global restrictions on the conformation of a peptide are possible by limiting the flexibility of the peptide strand through cyclization (Hruby el al., Biochem. J., 1990, 268: 249). For this purpose, amino acid side chains that are not involved in receptor recognition are bridged together or to the peptide backbone. Three representative examples are compounds wherein partial structures of each peptide are made into rings by linking two pennicillamine residues with a disulfide bridge (Mosberg et al., Proc. Natl. Acad. Sci. USA, 1983, 80: 5871), by formation of an amide bond between a lysine and an aspartate group (Charpentier et al., J. Med. Chem., 1989, 32: 1184), or by connecting two lysine groups with a succinate unit (Rodriguez et al., Int. J. Pept. Protein Res., 1990, 35: 441). These structures have been disclosed in the literature in the case of a cyclic enkephalin analog with selectivity for the .delta. opiate receptor (Mosberg et al., Proc. Natl. Acad. Sci. USA, 1983, 80:5871); or as agonists to the cholecystokinin-B receptor, found largely in the brain (Charpentier et al., J. Med. Chem., 1989, 32: 1184, Rodriguez et al., Int. J. Pept. Protein Res., 1990, 35: 441).
Another conceptual approach to the conformational constraint of peptides was introduced by Gilon et al. (Biopolymers, 1991, 31: 745) who proposed backbone to backbone cyclization of peptides. The theoretical advantages of this strategy include the ability to effect cyclization via the carbon or the nitrogen of the peptide backbone without interfering with side chains that may be crucial for interaction with a specific receptor of a given peptide. While the concept was envisaged as being applicable to any linear peptide of interest, in fact the limiting factor in the proposed scheme was the availability of suitable building units that must be used to replace the amino acids that are to be linked via bridging groups. The actual reduction to practice of the backbone cyclization concept was prevented by the inability to device a practical method of preparing suitable building units of amino acids other than glycine (Gilon et al., J. Org. Chem., 1992, 57: 5687). While analogs of other amino acids were attempted, the synthetic method used was unsuccessful or of a low yield as to preclude any general applicability.
Gilon et al. describe two basic approaches for the synthesis of suitable building units to produce building units for Boc and Fmoc chemistry peptide synthesis. For further details see, EP 564739A2 October, 1993; and Gilon et al. J. Org. Chem., 1992, 57: 5687). Both approaches deal with the reaction of a molecule of the general type X--CH(R)--CO--OR' (where X represents a leaving group which, in the example given, is either Br or Cl) with an amine which replaces the leaving group X. The amine bears the alkylidene chain which is terminated by another functional group, amine in the example described, which may or may not be blocked by a protecting group. In all cases the .alpha. nitrogen of the end product originates in a molecule which becomes the bridging chain for subsequent cyclization. This approach was chosen in order to take advantage of the higher susceptibility to nucleophilic displacement of a leaving group located next to the carboxylic group.
Peptoids are polimeric compounds formed by shifting amino acid side chains from the C.alpha. to the backbone nitrogen atom to yield N.alpha.-alkylated oligoglycine derivatives (Simon et al.,. Proc. Natl. Acad. Sci. USA, 1992, 89: 9367, WO 91/19735 (June, 1991) by Bartlett). Semipeptoids are polimeric compounds containing both N.alpha.-alkylated glycine derivatives and natural amino acids wherein the side chains are at C.alpha.. As referred herein in this document and especially in the claims section below, peptoid and semipeptoid analogs refer to chemical modifications such as but not limited to alkylation, hydroxylation dealkylation or dehydroxylation of one or more side chains at C.alpha. or N.alpha. of the peptoid or semipeptoid backbone. For further details regarding peptoids, semipeptoids and their chemistry the reader is referred to WO 91/19735 by Bartlett.
An important therapeutic advantage of peptoids as compared to peptides is their resistance to proteases (Miller et al., Med. Chem. Lett., 1994, 4: 2657; Miller et al., Drug Development Research, 1995, 35: 20). In addition, the peptoids approach permits sophisticated structure-function relationship studies using both natural (i.e., native, conventional) and unnatural (i.e., analog) side chains by simple chemistry. A few peptoids were already found to be biologically active (Simon et al., Proc. Natl. Acad. Sci. USA, 1992, 89: 9367; Zuckermann et al., J. Med. Chem., 1994, 37: 2678; Kessler, Angew. Chem. Int. Ed. Engl., 1993, 32: 543).
Oncogenic Ras is found in 40% of all cancers and is involved in over 90% of pancreatic tumors and over 50% of colon carcinomas (Gibbs et al., Cell, 1994, 77: 175). Thus, inhibition of the Ras function is believed to be a crucial target for cancer chemotherapy (Gibbs, Cell, 1991, 65: 1). Membrane localization of the oncogenic Ras is critical for its function and transformative potential (Kato et al., Proc. Natl. Acad. Sci. USA, 1992, 89: 6403). This membrane binding is achieved through a series of post translational modifications directed by its carboxy-terminal CAAX motive (SEQ ID NO:1) (where C is cysteine, A is an aliphatic residue and X is preferably serine or methionine). The first and most essential modification is farnesylation of the conserved cysteine residue, catalyzed by the enzyme farnesyltransferase. Subsequent modifications are dependent on its previous occurrence (Hancock, Current biology 1993, 3: 770). Inhibition of the farnesylation reaction either by site directed mutagenesis (Hancock et al., Cell, 1989, 57: 1167) or by synthetic farnesyltransferase inhibitors nullifies Ras membrane anchorage and reverses transformation by oncogenic Ras (Gibbs et al., Cell, 1994, 77: 175). Recent findings show that inhibition of farnesyltransferase by various CAAX (SEQ ID NO:1) peptidomimetics cause regression of ras-induced transformation is in whole cells (Cox et al., J. Biol. Chem., 1994, 269: 19203; Manne et al., Oncogene, 1995, 10: 1763; Patel et al., J. Med. Chem., 1995, 38: 435; Kohl et al., Science, 1993, 260: 1934; James et al., Science, 1993, 260: 1937) and in animals without significant toxic effects (Kohl et al., Proc. Natl. Acad. Sci. USA, 1994, 91: 9141; Kohl et al., Nature Medicine, 1995, 1: 792).
For example, the peptidomimetic compound L-739,749 (2(S)-[2(CH(CH.sub.3).sub.2)-amino-3-mercapto]-propylamino-3(S)-methyl]pen tyloxy-3 phenylpropionyl methionine sulphone methyl ester, SEQ ID NO:2) is a farnesyl protein transferase inhibitor. This compound (Kohl et al., Proc. Natl. Acad. Sci. USA, 1994, 91: 9141) selectively blocks ras-dependent transformation of cells in culture, by suppressing the anchorage-independent growth of RatI cells transformed with viral H-ras. Compound L-739,749 described therein was also found to inhibit the anchorage-independent growth of human adenocarcinoma cell line PSN-1, which harbors altered K-ras and p53 genes, and to suppress the growth of tumors in nude mice.
Another compound, designed by Kohl et al. as L-744,832 (2(S)-[2(CH(CH.sub.3).sub.2)-amino-3-mercapto]-propylamino-3(S)-methyl]pen tyloxy-3-phenylpropionyl methionine sulphone isopropyl ester, SEQ ID NO:3) is the isopropyl ester derivative of compound L-739,750 (2(S)-[2(CH(CH.sub.3).sub.2)-amino-3-mercapto]-propylamino-3(S) methyl]pentyloxy-3-phenylpropionyl methionine sulphone, SEQ ID NO:4), mimics the CAAX motif (SEQ ID NO:1) to which the famnesyl group is added during the post-translational modification process of Ras oncoprotein was found to be a potent and selective inhibitor of farnesyltransferase. In MMTV-v-Ha-ras transgenic mice bearing palpable tumors (Kohl et al., Nature Medicine, 1995, 1: 792), daily administration of compound L-744,832 caused tumor regression. These results suggest that, in some cancers, CAAX (SEQ ID NO:1) analog farnesyltransferase inhibitors may be used as anti tumor agents (Gibbs et al., Cell, 1994, 77: 175).